Introduction to Bioinformatics Databases

DNARNAphenotypeprotein Central dogma of molecular biology A main focus of bioinformatics is to study molecular sequence data to gain insight into a broad range of biological problems.

After Pace NR (1997) Science 276:734 Page 6 With the use of bioinformatics we can learn the variation that occur between species, and we can deduce the evolutionary history of life on Earth.

Growth of GenBank Base pairs of DNA (billions) Sequences (millions) December 1982 June 2006

Growth of the International Nucleotide Sequence Database Collaboration Base pairs of DNA (billions) Base pairs contributed by GenBank EMBL DDBJ

DNARNAprotein Central dogma of molecular biology genometranscriptomeproteome Central dogma of bioinformatics and genomics

DNARNA cDNA ESTs UniGene phenotype genomic DNA databases protein sequence databases protein Fig. 2.2 Page 20

GenBank EMBLDDBJ There are three major public DNA databases The underlying raw DNA sequences are identical Page 16

GenBankEMBLDDBJ Housed at EBI European Bioinformatics Institute There are three major public DNA databases Housed at NCBI National Center for Biotechnology Information Housed in Japan Page 16

>300,000 species are represented in GenBank Table 2-1

Taxonomy nodes at NCBI

The most sequenced organisms in GenBank Homo sapiens 10.7 billion bases Mus musculus 6.5b Rattus norvegicus 5.6b Danio rerio 1.7b Zea mays 1.4b Oryza sativa 0.8b Drosophila melanogaster0.7b Gallus gallus 0.5b Arabidopsis thaliana 0.5b Updated GenBank release Table 2-2 Page 18

The most sequenced organisms in GenBank Homo sapiens 11.2 billion bases Mus musculus 7.5b Rattus norvegicus 5.7b Danio rerio 2.1b Bos taurus1.9b Zea mays 1.4b Oryza sativa (japonica) 1.2b Xenopus tropicalis0.9b Canis familiaris0.8b Drosophila melanogaster0.7b Updated GenBank release Table 2-2 Page 18

The most sequenced organisms in GenBank Homo sapiens 12.3 billion bases Mus musculus 8.0b Rattus norvegicus 5.7b Bos taurus3.5b Danio rerio 2.5b Zea mays 1.8b Oryza sativa (japonica) 1.5b Strongylocentrotus purpurata1.2b Sus scrofa1.0b Xenopus tropicalis1.0b Updated GenBank release Table 2-2 Page 18

National Center for Biotechnology Information (NCBI) Page 24

Types of Data in GenBank  DNA level  RNA level (cDNA)  Protein sequences.  …

Fig. 2.5 Page 25

Fig. 2.5 Page 25

PubMed is… National Library of Medicine's search service 16 million citations in MEDLINE links to participating online journals PubMed tutorial (via “Education” on side bar) Page 24

Entrez integrates… the scientific literature; DNA and protein sequence databases; 3D protein structure data; population study data sets; assemblies of complete genomes Page 24

Entrez is a search and retrieval system that integrates NCBI databases Page 24

BLAST is… Basic Local Alignment Search Tool NCBI's sequence similarity search tool supports analysis of DNA and protein databases 100,000 searches per day Page 25

OMIM is… Online Mendelian Inheritance in Man catalog of human genes and genetic disorders edited by Dr. Victor McKusick, others at JHU Page 25

Books is… searchable resource of on-line books Page 26

TaxBrowser is… browser for the major divisions of living organisms (archaea, bacteria, eukaryota, viruses) taxonomy information such as genetic codes molecular data on extinct organisms Page 26

Structure site includes… Molecular Modelling Database (MMDB) biopolymer structures obtained from the Protein Data Bank (PDB) Cn3D (a 3D-structure viewer) vector alignment search tool (VAST) Page 26

Accessing information on molecular sequences Page 26

Accession numbers are labels for sequences NCBI includes databases (such as GenBank) that contain information on DNA, RNA, or protein sequences. You may want to acquire information beginning with a query such as the name of a protein of interest, or the raw nucleotides comprising a DNA sequence of interest. DNA sequences and other molecular data are tagged with accession numbers that are used to identify a sequence or other record relevant to molecular data. Page 26

What is an accession number? An accession number is label that used to identify a sequence. It is a string of letters and/or numbers that corresponds to a molecular sequence. Examples (all for retinol-binding protein, RBP4): X02775GenBank genomic DNA sequence NT_030059Genomic contig Rs dbSNP (single nucleotide polymorphism) N An expressed sequence tag (1 of 170) NM_006744RefSeq DNA sequence (from a transcript) NP_007635RefSeq protein AAC02945GenBank protein Q28369SwissProt protein 1KT7Protein Data Bank structure record protein DNA RNA Page 27

Four ways to access DNA and protein sequences [1] Entrez Gene with RefSeq [2] UniGene [3] European Bioinformatics Institute (EBI) and Ensembl (separate from NCBI) [4] ExPASy Sequence Retrieval System (separate from NCBI) Page 27

4 ways to access protein and DNA sequences [1] Entrez Gene with RefSeq Entrez Gene is a great starting point: it collects key information on each gene/protein from major databases. It covers all major organisms. RefSeq provides a curated, optimal accession number for each DNA (NM_006744) or protein (NP_007635) Page 27

From the NCBI home page, type “rbp4” and hit “Go” revised Fig. 2.7 Page 29

revised Fig. 2.7 Page 29

By applying limits, there are now just two entries

revised Fig. 2.8 Page 30 Entrez Gene (top of page) Note that links to many other RBP4 database entries are available

Entrez Gene (middle of page)

Entrez Gene (bottom of page)

Fig. 2.9 Page 32

Fig. 2.9 Page 32

Fig. 2.9 Page 32

FASTA format Fig Page 32

FASTA format

What is an accession number? An accession number is label that used to identify a sequence. It is a string of letters and/or numbers that corresponds to a molecular sequence. Examples (all for retinol-binding protein, RBP4): X02775GenBank genomic DNA sequence NT_030059Genomic contig Rs dbSNP (single nucleotide polymorphism) N An expressed sequence tag (1 of 170) NM_006744RefSeq DNA sequence (from a transcript) NP_007635RefSeq protein AAC02945GenBank protein Q28369SwissProt protein 1KT7Protein Data Bank structure record protein DNA RNA Page 27

NCBI’s important RefSeq project: best representative sequences RefSeq (accessible via the main page of NCBI) provides an expertly curated accession number that corresponds to the most stable, agreed-upon “reference” version of a sequence. RefSeq identifiers include the following formats: Complete genomeNC_###### Complete chromosomeNC_###### Genomic contigNT_###### mRNA (DNA format)NM_###### e.g. NM_ ProteinNP_###### e.g. NP_ Page 29-30

Accession MoleculeNote AP_ Protein Protein products; alternate NC_ Genomic Complete genomic molecules NG_ Genomic Incomplete genomic regions NM_ mRNATranscript products; mRNA NM_ mRNATranscript products; 9-digit NP_ ProteinProtein products; NP_ ProteinProtein products; 9-digit NR_ RNANon-coding transcripts NT_ GenomicGenomic assemblies NW_ GenomicGenomic assemblies NZ_ABCD GenomicWhole genome shotgun data XM_ mRNATranscript products XP_ ProteinProtein products XR_ RNATranscript products YP_ ProteinProtein products ZP_ Protein Protein products NCBI’s RefSeq project: accession for genomic, mRNA, protein sequences

Ensembl to access protein and DNA sequences Try Ensembl at for a premier human genome web browser. Ensembl is a joint scientific project between the European Bioinformatics InstituteEuropean Bioinformatics Institute and the Wellcome Trust Sanger InstituteWellcome Trust Sanger Institute, Its aim is to provide a centralised resource for geneticists, molecular biologists and other researchers studying the genomesgenomes of our own species and other vertebrates.vertebrates We will encounter Ensembl as we study the human genome, BLAST, and other topics.

click human

Species in Ensembl FISHES BIRDS REPTILES MAMMALS PLACENTALS MONOTREMES MARSUPIALS OTHER BIRDS PALEOGNATHS PASSERINES CROCODILES TURTLES LIZARDS AMPHIBIANS TELEOSTS SHARKS RAYS LATIMERIA BICHIR/POLYPTERUS LUNGFISHES AGNATHANS NON-VERTEBRATES

enter RBP4

Five ways to access DNA and protein sequences [1] Entrez Gene with RefSeq [2] UniGene [3] European Bioinformatics Institute (EBI) and Ensembl (separate from NCBI) [4] ExPASy Sequence Retrieval System (separate from NCBI) Page 33

ExPASy to access protein and DNA sequences ExPASy sequence retrieval system (ExPASy = Expert Protein Analysis System) Visit Page 33

Fig Page 33

Example of how to access sequence data: HIV-1 pol There are many possible approaches. Begin at the main page of NCBI, and type an Entrez query: hiv-1 pol Page 34

Searching for HIV-1 pol: Following the “genome” link yields a manageable three results

Example of how to access sequence data: HIV-1 pol For the Entrez query: hiv-1 pol there are about 40,000 nucleotide or protein records (and >100,000 records for a search for “hiv-1”), but these can easily be reduced in two easy steps: --specify the organism, e.g. hiv-1[organism] --limit the output to RefSeq! Page 34

only 1 RefSeq over 100,000 nucleotide entries for HIV-1

Examples of how to access sequence data: histone query for “histone”# results protein records21847 RefSeq entries7544 RefSeq (limit to human)1108 NOT deacetylase697 At this point, select a reasonable candidate (e.g. histone 2, H4) and follow its link to Entrez Gene. There, you can confirm you have the right gene/protein

Access to Biomedical Literature Page 35

PubMed at NCBI to find literature information

PubMed is the NCBI gateway to MEDLINE. MEDLINE contains bibliographic citations and author abstracts from over 4,600 journals published in the United States and in 70 foreign countries. It has >14 million records dating back to Page 35

PubMed search strategies Try the tutorial (“education” on the left sidebar) Use boolean queries (capitalize AND, OR, NOT) lipocalin AND disease Try using “limits” Try “Links” to find Entrez information and external resources Obtain articles on-line via Welch Medical Library (and download pdf files): Page 35

lipocalin AND disease (60 results) lipocalin OR disease (1,650,000 results) lipocalin NOT disease (530 results) 1 AND 2 1 OR 2 1 NOT Fig Page 34 8/04